Throughout this application, various publications are referenced in parentheses by author and year. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains.
Second harmonic generation (SHG) is a powerful spectroscopic tool for studying interfacial regions at the molecular scale, but to date has been confined mainly to non-biological systems. Recently, however, SHG has been extended to the study of a SH-active moiety at a supported lipid membrane system (Salafsky and Eisenthal, 2000a), a useful model for biological studies, and to the detection of protein adsorption at charged interfaces through the indirect effect the protein has on polarized water molecules near the surface (Salafsky and Eisenthal, 2000b). Direct detection of molecules such as proteins at interfaces could be useful in a number of biological studies, for example in studies of protein-receptor binding at a membrane or cellular interface or in the development of biosensors, but is hindered by the intrinsically low SH cross-section of proteins. For detection of molecules by SHG, the SH-active moiety must possess a hyperpolarizability and a net orientation at the interface. Although some proteins do contain chromophoric cofactors which are SH-active, their absorption is usually quite low or they are centrosymmetric. Other sources of SH activity in proteins include the aromatic amino acid side chains which are weakly SH-active. However, their varying orientations within the protein would reduce any SH signal.
The present application discloses the concept and technique of a ‘SHG-label’. SHG labels are second harmonic-active moieties which can be attached to a molecule or particle of interest that is not SH-active or only weakly SH-active, in order to render the molecule amenable to study at an interface. The labeled molecules may then be studied by surface-selective techniques such as second harmonic generation or sum-frequency generation. The technique can be illustrated by covalently labeling a protein, cytochrome c, with a SH-active moiety which is specific for either amine or sulfhydryl groups, common chemical moieties which exist on the surface of many protein molecules as part of their amino acid side-chains. Unlike detection with fluorescent labels, SHG-labels have the important advantage that only labeled proteins at an interface, and with a net orientation, contribute to the second harmonic signal; labeled protein molecules in the bulk contribute no signal. Furthermore, unlabeled molecules at the interface are undetectable. SHG-labels should find use in a variety of biological applications including studies of protein-protein, protein-membrane, and cell-cell interactions. SHG-labels can also be used to study other systems such as nanoparticle surfaces and polymer systems (polymer beads). In turn the labelled nanoparticle or labelled polymer bead can be used for example as a sensor of molecules in the surrounding medium.